recent, independent and anthropogenic origins of trypanosoma...

Recent, Independent and Anthropogenic Origins ofTrypanosoma cruzi HybridsMichael D. Lewis1*, Martin S. Llewellyn1, Matthew Yeo1, Nidia Acosta1,2, Michael W. Gaunt1, Michael A.

Miles1

1 Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom,

2 Departamento de Medicina Tropical, Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, Asunción, Paraguay

Abstract

The single celled eukaryote Trypanosoma cruzi, a parasite transmitted by numerous species of triatomine bug in theAmericas, causes Chagas disease in humans. T. cruzi generally reproduces asexually and appears to have a clonal populationstructure. However, two of the six major circulating genetic lineages, TcV and TcVI, are TcII-TcIII inter-lineage hybrids that arefrequently isolated from humans in regions where chronic Chagas disease is particularly severe. Nevertheless, a prevalentview is that hybridisation events in T. cruzi were evolutionarily ancient and that active recombination is of littleepidemiological importance. We analysed genotypes of hybrid and non-hybrid T. cruzi strains for markers representingthree distinct evolutionary rates: nuclear GPI sequences (n = 88), mitochondrial COII-ND1 sequences (n = 107) and 28polymorphic microsatellite loci (n = 35). Using Maximum Likelihood and Bayesian phylogenetic approaches we dated keyevolutionary events in the T. cruzi clade including the emergence of hybrid lineages TcV and TcVI, which we estimated tohave occurred within the last 60,000 years. We also found evidence for recent genetic exchange between TcIII and TcIV andbetween TcI and TcIV. These findings show that evolution of novel recombinants remains a potential epidemiological risk.The clearly distinguishable microsatellite genotypes of TcV and TcVI were highly heterozygous and displayed minimal intra-lineage diversity indicative of even earlier origins than sequence-based estimates. Natural hybrid genotypes resembledtypical meiotic F1 progeny, however, evidence for mitochondrial introgression, absence of haploid forms and previousexperimental crosses indicate that sexual reproduction in T. cruzi may involve alternatives to canonical meiosis. Overall, thedata support two independent hybridisation events between TcII and TcIII and a recent, rapid spread of the hybrid progenyin domestic transmission cycles concomitant with, or as a result of, disruption of natural transmission cycles by humanactivities.

Citation: Lewis MD, Llewellyn MS, Yeo M, Acosta N, Gaunt MW, et al. (2011) Recent, Independent and Anthropogenic Origins of Trypanosoma cruzi Hybrids. PLoSNegl Trop Dis 5(10): e1363. doi:10.1371/journal.pntd.0001363

Editor: Jane M. Carlton, New York University, United States of America

Received January 19, 2011; Accepted August 31, 2011; Published October 11, 2011

Copyright: � 2011 Lewis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the BBSRC (http://www.bbsrc.ac.uk/), the Wellcome Trust (http://www.wellcome.ac.uk/) and the European Union SeventhFramework Programme (http://cordis.europa.eu/fp7) grant 223034 (‘‘ChagasEpiNet’’). The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Trypanosoma cruzi is a single celled eukaryotic parasite, which is

transmitted to vertebrate hosts via the faeces of blood-sucking

triatomine bugs. It is the aetiological agent of Chagas disease in

humans, which results in the death of ,13,000 people and the lossof 649,000 disability-adjusted life years (DALYs) per year [1].

Transmission can also occur congenitally or through contaminat-

ed blood products and organs. T. cruzi is currently split into six

genetic lineages or discrete typing units (DTUs), previously named

TcI, TcIIa, IIb, IIc, IId and IIe [2,3] but recently revised by broad

consensus to TcI, TcIV, TcII, TcIII, TcV and TcVI respectively

[4]. Analysis of multiple molecular markers shows that a lack of

inter-lineage recombination generally preserves the individuality of

each DTU [2,5–7]. Recent studies of large samples of TcI and

TcIII using highly variable microsatellite markers have shown that

clonal population structure, may persist at an intra-lineage level

[8]. However, recombination at the scale of active transmission

cycles is known to occur [9] and the failure to detect it more

frequently is potentially a result of high rates of inbreeding [10],

gene conversion [8] or insufficient sampling of infra-populations

[11].

Many pathogenic eukaryotic microorganisms have essentially

clonal population structures while also having non-obligate sexual

cycles, which may enable them to adapt to environmental changes

[12]. Although recombination may be rare in such pathogens, it

can lead to the evolution and spread of epidemiologically

important traits, including those relating to virulence, transmission

dynamics and drug resistance [13]. Indeed, recombination events

have shaped the evolution of at least some T. cruzi lineages.Comparison of nucleotide sequences showed that two lineages,

TcV and TcVI (which includes the genome strain CL Brener),

have arisen through hybridisation between genetically distinct

parents [14–17]. The identification of these lineages as hybrid was

unequivocal because they possess fully intact alleles from two other

DTUs (TcII and TcIII) that are so distinct that they could not

have arisen independently. It is not clear whether TcV and TcVI

are the products of independent hybridisation events [15] or a

single hybridisation event followed by clonal divergence [17].

Furthermore, the resolution afforded by the genetic markers used

www.plosntds.org 1 October 2011 | Volume 5 | Issue 10 | e1363

so far has been insufficient to resolve whether these hybridisation(s)

were evolutionarily ancient [14,18] or recent events [16,17]. The

ecological circumstances of the origin of TcV and TcVI are not

known. While 5S rDNA sequences suggested the TcII parent may

have originated in south-western South America [17], too few

TcIII isolates have been characterised sufficiently to permit similar

investigation of the other putative parent.

Generation of intra-lineage TcI hybrids in vitro proved that the

capacity for genetic exchange has been retained [19], however,

there are important differences between these experimental

hybrids and natural TcV and TcVI isolates [20]. Genetic

characterisation of the experimental hybrids indicated they were

formed by diploid-diploid fusion, forming a tetraploid intermedi-

ate, followed by genome erosion resulting in progeny with DNA

contents approximately 70% higher than the parental strains

[19,20]. TcV and TcVI strains, by contrast, have DNA contents

equivalent to those found in TcII and TcIII. The experimental

TcI hybrids inherited .2 alleles at multiple genetic loci [19],whereas TcV and TcVI typically have only one or two alleles per

locus [20]. Finally, uniparental inheritance of kDNA maxicircles

occurred in both natural and experimental hybrids [16,19]. The

cytological mechanism of hybridisation in natural T. cruzi

populations therefore remains undefined and is yet to be

reconciled with that documented for in vitro hybrids. However,

the discovery of conserved meiotic gene homologues in several

basally diverging protists, including kinetoplastids, Giardia andTrichomonas implies that the common ancestor of all extant

eukaryotes was capable of meiotic recombination [21,22].

In this study molecular markers from three sequence classes

(nuclear coding sequence, mitochondrial (kinetoplast maxicircle)

coding sequence, and multiple microsatellite loci), each with

inherently different mutation rates, were used to analyse the

origins and evolution of T. cruzi at several overlapping levels of

resolution. Recent advances in the capacity for large scale

multilocus microsatellite typing [8] permitted high resolution

analysis of both parental and hybrid lineages leading to novel

insights into the timing, ecological circumstances and cytological

mechanism of hybridisation in T. cruzi.

Materials and Methods

SamplesDNA samples for a core panel of 35 T. cruzi clones representing

hybrid lineages TcV and TcVI and parental lineages TcII and

TcIII were used for all analyses; up to 53 additional strains

representing all six lineages were used in selected analyses as

indicated (Tables 1 and S1). DTU-level genotypes were determined

using a previously described triple-marker assay [23]. The samples

were derived from diverse hosts and vectors from geographical

locations representative of the distribution of each lineage.

Nucleotide sequencesNucleotide sequences were determined for a fragment of glucose-

6-phosphate isomerase (GPI), a single copy nuclear gene, and for aregion of the kDNA maxicircle, spanning regions of two adjacent

genes, cytochrome oxidase subunit II (COII) and NADH dehydro-genase subunit 1 (ND1). PCR amplification of GPI was performed asdescribed previously [23]. PCR amplification reactions for COII-ND1 contained 16 quantity NH4 buffer, 0.2 mM of dNTPs,3.5 mM MgCl2, 1 pmol/ml of each primer, 1 Unit of Taq DNApolymerase (all Bioline, UK) and 10–100 ng genomic DNA using

primers ND1.3A and COII.2A [16]. Amplification conditions were

94uC for 3 mins, 30 cycles of 94uC for 30 s, 58uC for 1 min, 72uCfor 2 mins, followed by a final elongation step at 72uC for 10 mins.

Sequencing reactions were performed using the ABI Prism

BigDye3.1 terminator cycle sequencing kit (Applied Biosystems,

UK) using PCR primers and additional internal sequencing

primers in some cases: for GPI, primer GPI.1 [19]; for COII-ND1,primers COII.A400 [16], COII-INTL1 (59-CAAAAGATAA-TAACACTATAACAGAATC-39) and COII-INTL2 (59-CAAA-AGATAATAACACTATAACAG-39). For resolution of GPIhaplotypes, PCR products were cloned using the pGEM-T Easy

vector kit (Promega, UK) and inserts from up to 12 clones were

sequenced. Sequences for outgroups and 43 additional T. cruzistrains were obtained from GenBank; accession numbers are given

in Table S1.

Phylogenetic analysis of GPI and COII-ND1Sequences were aligned using CLUSTAL_X [24] and sequence

diversity statistics calculated using DnaSP v.5 [25]. Unique

haplotypes were identified and aligned with outgroup sequences

to produce final alignments. Each alignment was used to

reconstruct multiple alternative tree topologies in MEGA4 [26]

using (a) Neighbour Joining (NJ) using Kimura’s 2-parameter

(K2P) [27], Tamura-Nei (TN93) [28], and maximum composite

likelihood [29] substitution models and (b) Maximum Parsimony

(MP) using the close neighbour interchange procedure, which

generated 143 and 98 trees of equal length for GPI and COII-ND1respectively. Bootstrap support for clades was estimated using

1000 pseudo-replicate datasets. Maximum likelihood (ML) analysis

was conducted for each topology using PAML v.4 [30]

implementing Shimodaira-Hasegawa (SH) tests [31] to test for

statistically significant differences in likelihoods between topolo-

gies. The program FINDMODEL (http://www.hiv.lanl.gov/

content/sequence/findmodel/findmodel.html) was used to select

the most appropriate nucleotide substitution model for each data

set: GTR (REV)+gamma for GPI and TN93+gamma for COII-ND1. In each ML analysis a discrete gamma distribution modelwith four rate categories was applied to account for rate

heterogeneity among sites; the gamma distribution shape param-

Author Summary

Trypanosoma cruzi is a parasite that causes Chagas diseasein humans, an often fatal condition affecting at least 8million people. The clinical outcome of Chagas disease isvariable, which is probably partially attributable to geneticdifferences between T. cruzi strains. Differences between T.cruzi strains can arise by gradual accumulation ofmutations in independent lineages (clonal reproduction)or by recombination between strains, which generatesnew combinations of existing alleles (sexual reproduction).Although sex has been observed experimentally, andepidemiologically important T. cruzi subgroups (TcV TcVI)originated via hybridisation events, active recombination isconsidered to be extremely rare. Our aim was to determinewhen recombination events have occurred during theevolution of T. cruzi. We analysed differences within andbetween T. cruzi subgroups in DNA sequences that evolveat different speeds. We found multiple recombinationevents, several of which were very recent in evolutionaryterms. We show that TcV and TcVI most likely originated asa result of human activities that promoted mixing betweensubgroups, suggesting a continuing risk for emergenceand spread of new genotypes generated by sexualreproduction. We also found evidence that sexual repro-duction in T. cruzi could involve mechanisms different fromthose seen in multi-cellular eukaryotes.

Origins of T. cruzi Hybrids


eter, a, was estimated from the data. To test for rate constancy (i.e.a molecular clock) ML analysis was performed using topologies

rooted using either Trypanosoma brucei sequences (GPI) or T. cruzimarinkellei sequences (COII-ND1), with all branches constrained to asingle rate of evolution. The resulting likelihood was compared to

the unconstrained model using a likelihood ratio test (LRT).

Estimation of divergence timesBEAST v1.5.3 [32] was used to estimate divergence times. This

program implements a Bayesian Markov chain Monte Carlo

(MCMC) procedure to sample the posterior probability of trees

generated under a specified prior evolutionary model. One

advantage of this approach is that by sampling from a large

number of trees, the statistical credibility (the 95% highest

posterior density (HPD) interval) associated with parameter

estimates (e.g. mutation rates, divergence times) can be obtained,

thereby more accurately reflecting the uncertainties of phyloge-

netic inference than analyses generating a single tree. For each

analysis the same nucleotide substitution model as implemented in

the ML analysis was used. For GPI we applied a strict molecular

Table 1. Genotypes for core samples analysed in the study.

GPI COII-ND1MLMT(19 loci)

MLMT(28 loci)

Haplotype code(s)

Strain DTU Location Host/Vector Allele 1 Allele 2 Haplotype code MLG code MLG code

Tu18 cl2 TcII Tupiza, BO Triatoma infestans Hap nG-21 Hap nG-22 Hap kCN-26 19/B01 28/B01

Rita cl5 TcII Bahia, BR Homo sapiens Hap nG-21 Hap kCN-27 19/B02 28/B02

CBB cl2 TcII Region IV, CL Homo sapiens Hap nG-21 Hap nG-22 Hap kCN-27 19/B03 28/B03

Pot7a cl1 TcII Boqueron, PY Triatoma infestans Hap nG-21 Hap kCN-27 19/B04 28/B04

Pot7b cl5 TcII Boqueron, PY Triatoma infestans Hap nG-21 Hap kCN-27 19/B04 28/B04

IVV cl4 TcII Cuncumen, CL Homo sapiens Hap nG-21 Hap nG-22 Hap kCN-26 19/B05 28/B05

Chaco23 col4 TcII Pr.Hayes, PY Triatoma infestans Hap nG-21 Hap nG-22 Hap kCN-25 19/B06 28/B06

Esm cl3 TcII Bahia, BR Homo sapiens Hap nG-21 Hap kCN-27 19/B04 28/B04

T665 cl1 TcII Pr.Hayes, PY Triatoma infestans Hap nG-22 Hap kCN-28 19/B07 28/B07

ARMA13 cl1 TcIII Boqueron, PY Dasypus novemcinctus Hap nG-14 Hap kCN-15 19/C01 28/C01

JA2 cl2 TcIII Amazonas, BR Monodelphis sp. Hap nG-15 Hap kCN-14 19/C02 28/C02

SABP19 cl1 TcIII Vitor, PE Triatoma infestans Hap nG-16 Hap nG-17 Hap kCN-22 19/C03 28/C03

ARMA18 cl3 TcIII Boqueron, PY Dasypus novemcinctus Hap nG-14 Hap nG-18 Hap kCN-13 19/C04 28/C04

CM25 cl2 TcIII Carimaga, CO Dasyprocta fugilinosa Hap nG-15 Hap nG-19 Hap kCN-13 19/C05 28/C05

M5631 cl5 TcIII Para, BR Dasypus novemcinctus Hap nG-15 Hap nG-20 Hap kCN-18 19/C06 28/C06

M6241 cl6 TcIII Para, BR Homo sapiens Hap nG-15 Hap nG-20 Hap kCN-17 19/C07 28/C07

85/847 cl2 TcIII Alto Beni, BO Dasypus novemcinctus Hap nG-15 Hap kCN-11 19/C08 28/C08

X9/3 TcIII Pr. Hayes, PY Canis familiaris Hap nG-14 Hap kCN-22 19/C09 28/C09

X109/2 TcIII Pr.Hayes, PY Canis familiaris Hap nG-16 Hap kCN-22 19/C10 28/C10

Sc43 cl1 TcV Santa Cruz, BO Triatoma infestans Hap nG-22 Hap nG-23 Hap kCN-19 19/D01 28/D01

Para6 cl4 TcV Paraguari, PY Triatoma infestans Hap nG-22 Hap nG-23 Hap kCN-20 19/D01 28/D01

92-80 cl2 TcV Santa Cruz, BO Homo sapiens Hap nG-22 Hap nG-23 Hap kCN-21 19/D01 28/D01

Para4 cl3 TcV Paraguari, PY Triatoma infestans Hap nG-22 Hap nG-23 Hap kCN-20 19/D01 28/D01

Chaco2 cl3 TcV Boqueron, PY Triatoma infestans Hap nG-22 Hap nG-23 Hap kCN-20 19/D01 28/D01

PAH179 cl5 TcV Chaco, AR Homo sapiens Hap nG-22 Hap nG-23 Hap kCN-20 19/D02 28/D02

Vinch101 cl1 TcV Limari, CL Triatoma infestans Hap nG-22 Hap nG-23 Hap kCN-20 19/D01 28/D01

Bug2148 cl1 TcV Rio Gr. do Sul, BR Triatoma infestans Hap nG-22 Hap nG-23 Hap kCN-20 19/D01 28/D01

CL Brener TcVI Rio Gr. do Sul, BR Triatoma infestans Hap nG-22 Hap nG-14 Hap kCN-24 19/E01 28/E01

VFRA1 cl1 TcVI Francia, CL Triatoma infestans Hap nG-22 Hap nG-14 Hap kCN-24 19/E01 28/E01

Chaco17 col1 TcVI Pr. Hayes, PY Triatoma infestans Hap nG-22 Hap nG-14 Hap kCN-24 19/E01 28/E01

Tula cl2 TcVI Tulahuen, CL Homo sapiens Hap nG-22 Hap nG-14 Hap kCN-24 19/E02 28/E02

P251 cl7 TcVI Cochabamba, BO Homo sapiens Hap nG-22 Hap nG-14 Hap kCN-24 19/E03 28/E03

LHVA cl4 TcVI Chaco, AR Triatoma infestans Hap nG-22 Hap nG-14 Hap kCN-24 19/E01 28/E01

EPV20-1 cl1 TcVI Chaco, AR Triatoma infestans Hap nG-22 Hap nG-14 Hap kCN-24 19/E02 28/E02

Chaco9 col15 TcVI Pr. Hayes, PY Triatoma infestans Hap nG-22 Hap nG-14 Hap kCN-23 19/E01 28/E01

DTU, discrete typing unit; MLMT, multi-locus microsatellite typing; MLG, multi-locus genotype; Hap, Haplotype; BO, Bolivia; BR, Brazil; CL, Chile; PY, Paraguay; PE, Peru;CO, Colombia; AR, Argentina.doi:10.1371/journal.pntd.0001363.t001



clock and for COII-ND1 a relaxed clock with uncorrelated log-normally distributed rates [33]. A constant population coalescent

prior was used as the demographic model. Random tree topologies

were used as initial trees. To calibrate the rate estimation for GPIwe applied a normally distributed prior on the divergence time

between T. brucei sequences and T. cruzi clade sequences, i.e. theage of the root of the tree, with a mean of 100 million years ago

(MYA) and SD of 2.0, according to the well supported evidence for

the divergence of these groups concomitant with the separation of

the African and South American land masses [34]. For COII-ND1,

calibration was achieved in the same way but with a time applied

to the T. cruzi – T. cruzi marinkellei ancestral node (the root) with a

mean of 6.511 MYA and SD of 1.17, as inferred from the GPIresults. For each data set two MCMC chains of 16107 iterationswere run, with parameters logged every 1000 iterations and

removal of the first 10% of states as burn-in. Log-files were

checked for sufficient effective sampling sizes (ESSs) and

convergence of chains on similar posterior distributions using

TRACER v1.5 [35].

Microsatellite analysisGenotypes were obtained for the core 35 samples at 28

microsatellite loci and for an additional 46 strains at a subset of 19

microsatellite loci using previously described protocols [8,20];

physical positions and primer details are given in Table S2.

Microsatellite genotypes for some strains at some loci were from

previously published datasets as indicated [8,20,36]. The geno-

types were used to infer a measure of genetic distance between all

possible pairs of strains (pairwise distance [DAS]) under theassumptions of the infinite-alleles model (IAM) [37] as previously

[8]. Multi-allelic genotypes ($3 alleles per locus), observed for 7 of1826 genotypes, were treated as missing data. For each DTU,

heterozygosity indices were calculated in ARLEQUIN 3.0 [38].

Allelic richness (Ar) was used as a sample size-corrected measure of

diversity and was calculated in FSTAT [39]. In hybrid groups

TcV and TcVI, genotypes at individual loci were classed as hybrid

(TcII/TcIII) or non-hybrid (TcII/TcII or TcIII/TcIII) based on

the presence or absence of alleles in the TcII and TcIII parental

groups. In order to minimize the possibility of null alleles affecting

classification of TcV/VI genotypes, all instances of homozygosity

in these samples were confirmed across three replicates. For

inference of geographical relationships between TcV/VI hybrid

and TcII/III parent DTUs, the genetic identity between each

hybrid multilocus genotype (MLG) and all parent MLGs was

calculated using 1-DAS. Then for TcV and TcVI separately, the

mean identities with parental MLGs from countries with $2measurements (Brazil, Bolivia, Chile and Paraguay) were calcu-

lated and compared using t-tests.

Results

GPI nucleotide sequenceSequences for GPI were obtained for 86 T. cruzi strains allowing

a gap-free alignment of 1038 nucleotides to be assembled.

Heterozygous strains were found in all DTUs: TcI (n = 19/29),

TcII (n = 4/9), TcIII (n = 8/25), TcIV (n = 1/7), TcV (n = 8/8)

and TcVI (n = 8/8). A total of 34 distinct haplotypes and 58

polymorphic sites (5.59%) were identified (Tables 1 and S1).

Comparing cloned and uncloned sequence reads we classified 39/

247 (15.8%) as mosaics, most likely chimaeric products of PCR-

mediated recombination [40,41]. Once these were discarded two

haplotypes per strain with mutually exclusive single nucleotide

(SNP) patterns and compatible with the uncloned sequences were

consistently identifiable.

Sequence analysis (Figure 1) identified four major clades of T.cruzi sequences, equating to the T. cruzi lineages TcI, TcII, TcIIIand TcIV. Each TcV and TcVI strain had one TcII-derived and

one TcIII-derived haplotype. Three of these clades (TcI, II and

III) had robust bootstrap support. Sequences from North

American (NA) and South American (SA) TcIV strains were

clearly divergent and monophyly of these taxa was only

moderately supported. Examination of mean intra-lineage pair-

wise genetic distances showed that in our sample of strains the

predominantly domestic lineages TcII, TcV and TcVI were much

more homogeneous than strains from the predominantly sylvatic

lineages TcI, TcIII and TcIV. TcIII and TcIV sequences

contained fixed SNPs that were otherwise restricted to TcI or

TcII, as well as multiple lineage specific polymorphisms.

All TcV and TcVI strains shared the same intact TcII

haplotype (haplotype nG-22), which was also present in 5/9 TcII

isolates (Tu18 cl2, CBB cl2, IVV cl4, T665 cl1 and Chaco23 col4)

(Table 1). The low diversity in the TcII clade made it difficult to

infer geographical associations between the present isolates and

the TcII parent of TcV and TcVI but it is noteworthy that

haplotype nG-22 was not found in either of the two isolates from

Northern Brazil (Esm and Rita). The TcIII clade haplotype found

in all TcVI strains (haplotype nG-14) was also found in 13/25

of the TcIII isolates (Table 1). A different TcIII haplotype

(haplotype nG-23) was found in all TcV isolates but not in any

other DTU. However, haplotype nG-23 and haplotype nG-14

were closely related, with only one distinguishing SNP (834C/G).

There was evidence for some substructure within the TcIII clade,

with separation between a ‘Southern’ group [36] containing all

strains from Paraguay (11/11), Peru (1/1) and a subset of those

from Bolivia (4/7) and a ‘Northern’ group [36], containing strains

from Brazil (3/3), Colombia (2/2), Venezuela (1/1), and the

remainder from Bolivia (3/7). The haplotypes from the hybrids

TcV and TcVI were both unambiguously in the ‘Southern’ group

(Figure 1).

COII-ND1 nucleotide sequenceMitochondrial COII-ND1 sequences were obtained for 105 T.

cruzi strains allowing an alignment of 1118 nucleotides to beassembled. Thirty-nine unique haplotypes were identified with 198

polymorphic sites (17.7%) (Tables 1 and S1). Numerous small

indels (1–3 nt) were identified, as well as some larger indels,

including a large deletion of 245 nt (positions 671–915) in

haplotype kCN-26, previously found in Tu18 cl2 [16], and which

we also found in IVV cl4. Intra-lineage nucleotide diversities

(Table S3) revealed that hybrid groups TcV and TcVI had very

low genetic diversity. All TcV sequences were identical bar private

SNPs in Sc43 cl1 (position 818) and 92-80 cl2 (position 1073).

Likewise, all TcVI had identical sequences bar a private SNP in

Chaco9 col15 (position 1025). The mean genetic distance between

TcV and TcVI sequences was low (0.43%), but fixed inter-lineage

SNPs were found at three positions (105, 629 and 671).

Four major clades were recovered: (i) TcI, (ii) TcIV(NA)+threeTcI(NA), (iii) TcIII+TcV+TcVI+TcIV(SA), (iv) TcII (Figure 2).Within TcI four well-supported sub-clades corresponded broadly

to different geographical regions: first, all TcI sequences from

Central America (5/6), Colombia (2/2) and a subset of those from

Venezuela (2/3) and USA (2/5); second, sequences from South of

the Amazon (Bolivia (4/7), Chile (2/2), Peru (4/4) and southern

Brazil (3/3)); third, sequences from northern Brazil (5/5) and

Venezuela (1/3); and fourth, sequences from French Guyana (2/2)

and from a subset of isolates from Bolivia (3/7). No clear

correlations between different clades and different host species or

transmission cycles were apparent.



As for GPI, COII-ND1 sequences showed clear divergence

between North and South American TcIV strains. Moreover,

TcIV(SA) strains were paraphyletic and interspersed between

TcIII haplotypes. There were three separate instances of sharing

of identical or nearly identical mitochondrial haplotypes between

TcIV strains and strains from other DTUs (TcIII or TcI) for which

nuclear GPI sequences were highly divergent (Figures 1 and 2).

Such gross incongruence between mitochondrial and nuclear

phylogenies is likely indicative of historical genetic exchange events

resulting in mitochondrial introgression between DTUs.

The TcV and TcVI COII-ND1 sequences and TcIII COII-ND1

haplotype kCN-22 were more closely related to each other than to

any other TcIII sequence, forming a very strongly supported

monophyletic group. Haplotype kCN-22 differed from the TcV

and TcVI consensus sequences at only three or one nucleotide

position(s) respectively. By comparison, the next most similar TcIII

isolates had fixed differences at twelve positions compared to TcVI

or thirteen positions compared to TcV. Haplotype kCN-22 was

found in 6/18 TcIII strains: one human isolate from southern Peru

(SABP19 cl1), three were isolated from domestic dogs in the

Paraguayan Chaco (X9/3, X110/8, X109/2) [42], and two were

isolated from sylvatic mammals, again from the Paraguayan

Chaco (Sp4, Sp16) [36,43].

Estimation of divergence timesFor GPI sequences, rooted using T. brucei, the molecular clock

model was not significantly worse than the unconstrained model

(LRT p = 0.83; Table 2). Thus we used a time-calibrated strict

Figure 1. Maximum likelihood tree of GPI sequences (unique haplotypes). Four major clades are indicated according to corresponding T.cruzi lineage; TcV and TcVI haplotypes are within the parental TcII and TcIII clades from which they are derived. Where clear geographic associationswere evident these are indicated: US (samples from USA); SA (samples from South America); ‘Northern’ and ‘Southern’ groups within TcIII are asdefined previously [36]. Branches separating T. cruzi from T. rangeli and T. brucei (outgroup) are not shown. Bootstrap support values for branches (if.50%) are shown and were calculated for the equivalent nodes of the bootstrap consensus NJ/MP trees (1000 replicates). Scale units aresubstitutions/site.doi:10.1371/journal.pntd.0001363.g001



molecular clock in the Bayesian analysis of mutation rate and

divergence times for nodes in the GPI tree. For COII-ND1

sequences, rooted using T. cruzi marinkellei, the molecular clock was

rejected for the ML tree (LRT p = 0.032; Table 2). However, for

46/98 (46.9%) of the topologies tested the molecular clock was not

rejected (p.0.05) and none of these topologies gave likelihoodscores significantly worse than the ML tree (SH tests, p.0.05)

under the unconstrained model. Thus, instead of a strict molecular

clock we chose to apply a relaxed molecular clock model [33]

permitting rate variation across the tree to be factored into the

Bayesian estimation of divergence times.

The mean estimated mutation rates were 3.5561029 nucleotidesubstitutions/site/year (95% HPD limits 2.56–4.6061029) for GPIand 1.9461028 (95% HPD limits 1.05–3.0461028; rate coeffi-

Figure 2. Maximum likelihood tree of COII-ND1 sequences (unique haplotypes). Clades and subclades are indicated according tocorresponding major T. cruzi lineage (see text). Black circles indicate TcIII haplotypes and black triangles indicate TcIV haplotypes, clearly showingparaphyly of TcIV and TcIII sequences and sharing of identical haplotypes between subsets of TcIII and TcIV samples. Where clear geographicassociations were evident these are indicated using standard two letter country codes; TcIV(SA) are TcIV samples from South America. Bootstrapsupport values for branches (if .50%) are shown and were calculated for the equivalent nodes of the bootstrap consensus NJ/MP trees (1000replicates). Scale units are substitutions/site.doi:10.1371/journal.pntd.0001363.g002

Table 2. Molecular clock likelihood ratio tests.

Locus Haplotypes (n) Outgroup Model Clock? Log Ln 2*DLn (D) Ddf x2 p value

GPI 39 Unrooted GTR+G4 No 23236.663433

GPI 39 T. brucei GTR+G4 Yes 23251.047356 28.76785 37 0.831

COII-ND1 40 Unrooted TN93+G4 No 23151.651149

COII-ND1 40 T. cruzi marinkellei TN93+G4 Yes 23179.461322 55.62035 38 0.032

doi:10.1371/journal.pntd.0001363.t002



cient of variation = 0.34) for COII-ND1. Divergence time estimates

are given in Table 3. The time of the most recent common

ancestor (tMRCAs) for T. cruzi sensu stricto (i.e. excluding T. cruzi

marinkellei) was estimated at 3.35 MYA (GPI) or 4 MYA (COII-

ND1). For this and several other important nodes the two markers

yielded similar age estimates with overlapping 95% HPD limits,

including tMRCAs for TcI and TcII as well as the divergence of

North American and South American TcIV. The divergence of

TcI and TcIII COII-ND1 sequences, estimated at 2.44 MYA

(4.03–0.986), represents an estimate for the minimum age for a

founding hybridisation event between TcI and TcII, assuming

uniparental inheritance of the ancestral TcI maxicircle genome

[17]. We considered the possibility that if TcIII-VI strains are the

products of historical genetic exchange [17] then their inclusion

could introduce error into the dating estimates. However, we

found this not to be the case since data sets with the TcIII-VI

strains included and excluded yielded virtually identical estimates

of both substitution rates and divergence times (not shown). GPI

sequences did not provide sufficient resolution to date the

emergence of the hybrid DTUs TcV and TcVI but the more

variable maxicircle sequences supported a very recent origin with

tMRCAs estimated at 60,700 (944–126,000) years ago and 33,900

(523–91,300) years ago respectively.

MLMTWe applied MLMT to gain higher resolution of the genetic

diversity within and between hybrid and parental lineages. The 35

core TcII, TcIII, TcV and TcVI samples were typed at 28

polymorphic microsatellite loci (28 locus-analysis; Tables 1, S1 and

S4), of which eight were described previously for these strains [20].

Genotypes for additional TcI, TcIII and TcIV strains for 19 of

these loci were included for comparison in some cases (19-locus

analysis; Tables 1, S1 and S4). Unless additional sized peaks were

observed, microsatellite profiles were considered to represent

homozygous (one peak) or heterozygous (two peaks) diploid

genotypes. The majority of samples gave either one or two peak

sizes. Triple peak profiles were observed for CBB cl2 (TcII) at 6

loci: 6529(TA)b, 6529(CA)c, 6529(CA)a, 6529(TCC), (all linked on

a 17 kb region of chromosome 6), 10101(CA)c, (chromosome 27)

11283(TA)a (chromosome 40); DNA content analysis has shown

this strain is probably aneuploid [20]. The only other multi-allelic

profile was found for P251 cl7 (TcVI), which had three

10101(TA)/Set0 alleles (chromosome 27).

Pairwise genetic distances (DAS) were used to reconstructphylogenetic trees using the NJ method (Figure 3). Parental and

hybrid DTUs exhibited highly dissimilar patterns of genetic

diversity. Most TcII and TcIII strains had unique MLGs, with

high pairwise DAS genetic distances (.50%) and numerous privatealleles (Table 4; Figure 4). In contrast TcV and TcVI were

genetically uniform, with few, highly similar MLGs, low pairwise

genetic distances (2.2 and 1% respectively) and far fewer private

alleles (Table 4; Figure 4). Allelic richness (Ar) provided a measure

of genetic diversity independent of sample size and supported

markedly lower diversity in TcV/TcVI compared to TcII/TcIII

(Table 4). In the 19-locus analysis TcI, TcII, TcIII and TcIV all

showed similar patterns of high genetic differentiation between

strains with strong support for four monophyletic clusters

(Figure 3).

Hybrid and non-hybrid DTUs were also easily distinguishable

in terms of heterozygosity. TcV and TcVI were both highly

heterozygous (HO = 0.82/0.79). TcI and TcIII were far lessheterozygous (HO = 0.38/0.25). TcII displayed intermediate HO,and although this was close to overall HE, only 12/28 loci were inHardy-Weinberg equilibrium (not shown). Identical genotypes

were observed from geographically distant locations for domestic

isolates from TcII, TcV and TcVI (e.g. Esm cl3 from north-

eastern Brazil was identical to Pot7a cl1 and Pot7b cl5 from the

Paraguayan Chaco region).

Analysis of hybridisationFor many of the heterozygous loci in TcV and TcVI strains one

of the alleles was frequently observed in TcII strains while the

other allele was frequently observed in TcIII strains (Figures 4 and

S1). Inheritance through hybridisation and without subsequent

mutation is the most likely explanation, and was observed at too

many independent loci to attribute to homoplasy. Comparison of

the MLGs for TcV and TcVI also revealed fixed inter-DTU

genotypic differences at 75% (21/28) of microsatellite loci and of

the alleles that distinguished between TcV and TcVI 84% (37/44)

were present in parental DTU strains. Coupled with the striking

lack of intra-DTU diversity in both lineages this strongly suggests

the two DTUs are products of separate hybridisation events.

The pairwise DAS distances between each hybrid MLG andeach TcII (28 locus analysis) and TcIII (28 and 19 locus analysis)

sample were used to measure identity between members of each

parental DTU and each hybrid DTU. We then used these

distances to infer the approximate geographical location for the

origin of TcV and TcVI by grouping hybrid-parent distances

according to the country of origin of parental samples. This

revealed that TcV and TcVI genotypes were most closely related

to parental lineage strains from Chile and Paraguay respectively,

and furthermore, TcII/III strains from Brazil were significantly

less closely related to TcV/VI than those from Bolivia, Paraguay

or Chile (TcV only) (Figure 4B).

Table 3. Divergence times for selected species and T. cruzisubgroups.

Alignment Node/GrouptMRCA(MYA)

95% HPDLimits (MYA)

GPI T. brucei - T. cruzi (root) 99.9 104 - 96

T. cruzi - T. rangeli 31 39.3 - 22.9

T. cruzi - T. cruzi marinkellei 6.51 8.91 - 4.33

T. cruzi 3.35 4.76 - 2.06

TcI+TcIII–TcIV 2.24 3.17 - 1.38

TcI–TcIII 1.78 2.59 - 1.04

TcIV(NA) - TcIV(SA) 1.65 2.48 - 0.888

TcI 0.875 1.33 - 0.469

TcIII 0.827 1.32 - 0.353

TcIV(SA) 0.378 0.732 - 0.0968

TcII 0.221 0.543 - 0.00363

COII-ND1 T. cruzi - T. cruzi marinkellei(root)

6.04 8.47 - 3.69

T. cruzi 4 6.35 - 1.71

TcI–TcIII+IV+V+VI 2.44 4.03 - 0.986

TcIV(NA) - TcIII+IV(SA)+V+VI 0.847 1.46 - 0.317

TcI 0.517 0.911 - 0.184

TcII 0.148 0.303 - 0.0307

TcV+VI+Hap22 0.138 0.26 - 0.0336

TcV 0.0607 0.126 - 0.00944

TcVI 0.0339 0.0913 - 0.000523

doi:10.1371/journal.pntd.0001363.t003



The genotypes at each individual microsatellite locus in the

TcV/VI isolates were examined to predict the parental origin of

each allele given its presence in or absence from TcII/III isolates.

In this way the predicted ancestry of hybrid alleles could be

classified as (i) TcII, (ii) TcIII, (iii) TcII or TcIII, or (iv) unknown

(i.e. parent not sampled or de novo mutation) (Figures 4A and S1).

For TcV 65%, and for TcVI 58% of alleles were assigned to either

TcII or TcIII; either 33% (TcV) or 27% (TcVI) were found in

both TcII and TcIII; 2% (TcV) or 15% (TcVI) were found in

neither parental DTU. Based on this information there was

evidence of non-hybrid genotypes (i.e. both alleles derived from

the same parental DTU) at three loci in TcV: 6855(TTA)(GTT)

for one strain and 6789(TA) and 11283(TA)a for all strains; and

two loci in TcVI 6789(TA) and 11283(TA)a for all strains; in all

cases the genotypes comprised only TcIII-restricted alleles.

Discussion

Origin of T. cruziThe subgenus Schizotrypanum comprises approximately half a

dozen described trypanosome species, most of which evolved on

the South American continent after the break-up of Gondwana-

land, and are referred to as the ‘‘T. cruzi clade’’ [34,44]. By

analysing nuclear coding (GPI), mitochondrial coding (COII-ND1)

and nuclear non-coding (microsatellites) genetic markers, we were

able to resolve the evolutionary processes within this clade at

multiple, over-lapping levels of resolution. GPI sequences had the

slowest rate of evolution, estimated to have a nucleotide

substitution rate of 3.5561029 substitutions/site/year, reflectingits role as a house-keeping gene. The mitochondrial locus COII-

ND1 had a faster evolutionary rate, estimated at 1.8661028

substitutions/site/year on average, in keeping with the generally

higher rate of evolution in mtDNA than in nuclear DNA for

eukaryotes [45]. Microsatellites typically have mutation rates at

least three orders of magnitude higher than protein coding genes

[46]. Accordingly the microsatellite loci used in this study afforded

the highest resolution of intra-lineage diversity.

We estimated the dates of key events in the evolution of T. cruzi

and related species, including divergence of T. rangeli and T. cruzi

ancestral populations approximately 30 MYA, divergence of T.

cruzi s.s. and the Chiropteran-restricted subspecies T. cruzi

marinkellei at 6.5 – 6 MYA, and the tMRCA for T. cruzi s.s. at

4.0 – 3.35 MYA when the TcI and TcII lineages diverged. This

date closely coincides with the formation of the land bridge

between South and North America 3.5 – 3.1 MYA [47] suggesting

that T. cruzi diversification may have been influenced by the

subsequent changes in fauna known as the Great American

Interchange [48]. Previous estimates for the age of T. cruzi range

from 3–16 MYA, a date also obtained by calibration on the

breakup of Gondwanaland, and 10.6 MYA calibrated using a

universal rate of mutation of 1% MY21 for mitochondrial CYTB

[14]. TcIII and TcIV GPI sequences contained numerous SNPs

that were otherwise only found in TcI or TcII, as well as lineage-

specific polymorphisms. This pattern of SNPs is compatible with

an ancient hybridisation event between TcI and TcII as previously

suggested [17,49]. Such reticulate evolution explains the large

discrepancies between MP (based on individual sites) and NJ

(based on overall genetic distance) bootstrap values for relevant

nodes of the GPI phylogeny. The finding that the COII-ND1

Figure 3. Unrooted neighbour-joining microsatellite DAS treesshowing lack of diversity within hybrid lineages TcV and TcVI.A) Tree based on 28 loci typed across 35 samples from parental (TcII,TcIII) and hybrid (TcV, TcVI) lineages. B) Tree based on 19 loci typed

across 81 samples from all six major lineages TcI-VI. DAS-basedbootstrap values were calculated over 1000 trees from pseudo-replicatedatasets and those .70% are shown for the relevant nodes.doi:10.1371/journal.pntd.0001363.g003



sequences of TcI and TcIII/IV diverged an estimated 2.44 MYA

implies additional complexity because analysis of both COII-ND1

[16] and CYTB [14] shows there is far less mitochondrial diversity

both within and between TcIII and TcIV(SA) than would be

expected in light of the considerable divergence observed for

slower-evolving nuclear genes. This implies a mechanism acting to

homogenise maxicircle sequences while nuclear sequences remain

free to diverge. A partial explanation may stem from our finding of

multiple instances of clearly recent mitochondrial introgression

between TcIII and TcIV in South America, indicating additional

recombination events involving either substantial backcrossing or

kDNA exchange without exchange of nuclear material.

The sequence analysis showed clear divergence between North

and South American TcIV populations, estimated at 0.85–1.65

MYA, again most likely a result of faunal migrations between the

two continents. Migration of TcI from South to North America

may have occurred more recently as suggested by a strong

reduction in microsatellite diversity among North American TcI

isolates [8]. Additionally, this study builds on evidence [16]

supporting recent mitochondrial introgression between a subset of

TcI and TcIV strains in North America, which share almost

identical mitochondrial COII-ND1 sequences while displaying

extensive divergence for nuclear GPI sequences. Typing of further

nuclear loci is required to determine whether such events also

Figure 4. Patterns of microsatellite allelic inheritance in hybrid lineages TcV and TcVI. Based on analysis of 19 locus and 28 locus data setscombined. A) For each parental (TcII, TcIII) and hybrid (TcV, TcVI) group the charts show the proportion of microsatellite alleles that were also found ineach of the other three groups and the proportion of private alleles – those not found in any of the other three groups. B) Mean pairwise geneticidentities (1-DAS) between hybrid group (TcV and TcVI) MLGs and parental group (TcII and TcIII) MLGs from different geographical regions; DTU,discrete typing unit; error bars are S.E.M; brackets represent statistically significant 2-tailed t-tests with asterisks indicating: ** p,0.01, *** p,0.001.doi:10.1371/journal.pntd.0001363.g004

Table 4. Multilocus microsatellite summary statistics.

Data set DTU N/G Alleles Ho He MNA MNA SD Ar

Mean pairwise geneticdistance (DAS)

Mean pairwise geneticdistance (DAS) SD

28 loci TcII 9/7 110 0.640 0.644 3.929 1.184 3.670 0.501 0.264

TcIII 10/10 131 0.382 0.554 4.679 3.151 4.185 0.525 0.099

TcV 8/2 54 0.817 0.450 1.929 0.466 1.919 0.022 0.039

TcVI 8/2 52 0.786 0.424 1.857 0.525 1.821 0.010 0.009

19 loci TcI 26/26 85 0.235 0.454 4.474 2.855 3.341 0.429 0.106

TcIV 3/3 47 0.175 0.575 2.474 0.905 ND 0.701 0.105

TcII 9/7 71 0.601 0.600 3.737 1.327 3.548 0.472 0.251

TcIII 25/24 107 0.381 0.493 5.632 4.044 3.766 0.443 0.106

TcV 8/2 35 0.836 0.449 1.842 0.375 1.842 0.007 0.011

TcVI 8/3 36 0.842 0.456 1.895 0.459 1.895 0.014 0.014

DTU, discrete typing unit; N/G, number of samples/number of genotypes; Ho observed heterozygosity; He expected heterozygosity; MNA, mean number of alleles; ArAllelic richness; ND, not determined.doi:10.1371/journal.pntd.0001363.t004



involved transfer of any nuclear material, but the nuclear GPI andmicrosatellite data presented here indicate that it did not.

Our estimates of substitution rates and divergence times depend

on the prior assumption that T. brucei and T. cruzi diverged due tothe break-up of Gondwanaland ,100MYA. Biogeographic dataindicate this is a robust assumption. For example, Stevens et al

[34] showed that, with the exception of those associated with bats,

T. cruzi clade species are found exclusively in the New World andare most closely related to a kangaroo trypanosome. This is

consistent with the connection of South America to Australia via

Antarctica during the late Cretaceous [50]. Molecular data show

this biogeographic event also led to the evolution of the

Triatominae [51]. However, the recent finding of T. conorhini-likeand T. vespertilionis-like trypanosomes in two terrestrial mammals inWest Africa [52] illustrates the need for caution. In this case,

introduction of these trypanosomes from the New World seems a

plausible explanation given the capacity for host-switching in

trypanosomes and the well documented inter-continental dispersal

of T. cruzi clade species by bats (T. vespertilionis, T. dionisii, T. cruzimarinkellei) and rats (T. conorhini) [53].

Origin and clonal expansion of TcV and TcVIThe multilocus genotypes of the T. cruzi hybrid lineages TcV

and TcVI fit the expectations of relatively recent hybridisation:

they are highly heterozygous, exhibit minimal intra-lineage

diversity and have accumulated few, if any, private alleles by denovo mutation. This contrasts with TcI, TcII and TcIII, which aregenetically diverse, have abundant private alleles and generally

have lower than expected heterozygosity [8,36]. TcV and TcVI

have undergone dramatic clonal expansion: identical MLGs were

found in samples originating across a vast geographic area. Any

selfing or outcrossing would have produced homozygous geno-

types rather than the uniform heterozygosity observed at most loci.

The minority of TcV/VI microsatellite genotypes that were

homozygous can be explained by several phenomena, including

inheritance of identical alleles from both parents, loss of

heterozygosity (LOH) due to gene conversion, null alleles, or

non-meiotic inheritance (see below).

High resolution microsatellite data allowed us to address the

question of whether TcV and TcVI arose by a single event

followed by clonal diversification [17] or by separate hybridisation

events [15]. Our data support two independent hybridisation

events between distinct TcII and TcIII strains as the most

plausible scenario because they have fixed inter-lineage genotypic

differences at 75% of microsatellite loci and display virtually no

intra-lineage diversity. A single event leading to a hybrid

population from which TcV and TcVI are the only two surviving

lineages is unlikely. Firstly, the majority of microsatellite alleles

that distinguish TcV and TcVI were also present in parental

strains; independent inheritance is far more parsimonious than

repeated homoplasy. Secondly, TcV and TcVI have distinct GPIand COII-ND1 alleles, MLEE profiles [42,54,55], LSU rDNAsequences [23,56] and MLST profiles [57]; if these differences

were the result of divergence of TcV and TcVI from the same

ancestral hybrid population then a higher frequency of indepen-

dently derived (private) alleles at rapidly evolving microsatellite

loci would be expected. Alternatively, TcV and TcVI could have

evolved from distinct progeny from the same hybridisation event

between heterozygous TcII and TcIII parents i.e. siblings, with

different alleles inherited by the two hybrid lineages.

TcV and TcVI are almost exclusively found in domestic

transmission cycles and are the predominant genotypes in some

hyper-endemic regions such as the Gran Chaco area where severe

forms of Chagas disease are common [42,55,58–64]. Their clonal

reproductive mode and negligible genetic diversity have poten-

tially important biological and medical implications. For instance,

innate phenotypic variability for traits such as resistance to drugs

used to treat Chagas disease might be lower in TcV and TcVI

than in other, more diverse lineages. Equally, under certain

conditions the availability of diverse alleles may result in enhanced

fitness compared to either parental lineage (heterosis).

Geographic, temporal and ecological aspects ofhybridisation events

Fully intact or highly similar parental alleles identifiable in TcV

and TcVI permitted phylogenetic analysis to identify TcII and

TcIII strains most closely related to their actual parents. Nuclear

GPI sequences and microsatellite genotypes both supported asignificantly closer genetic relationship between TcII isolates from

Chile, Paraguay and Bolivia than with TcII from Brazil. This is

congruent with a study of 5S rDNA sequences [65], which linked

TcV/VI to TcII from Bolivia and Chile based on the presence of

an ‘invader sequence’ not found in TcII from Brazil. For the other

parental DTU, TcIII, GPI and microsatellites supported a closergenetic relationship with TcIII from Bolivia, Paraguay and Peru

than with TcIII from Brazil, Colombia or Venezuela. A previous

analysis of mitochondrial COII-ND1 sequences [16] showed thatthree TcIII isolates isolated from domestic dogs in the Paraguayan

Chaco were most similar to TcV/VI. We identified the same

COII-ND1 sequence in a human isolate from southern Peru and intwo Paraguayan Chaco sylvatic isolates. Overall, these data

support the Gran Chaco and adjoining Andean valleys in south-

western South America as the most probable origin of TcV and

TcVI, with mitochondrial sequences pointing more specifically to

the Paraguayan Chaco. Further analysis using additional strains

and nuclear coding loci that afford sufficient resolution will be

required to confirm this hypothesis.

This leads to the questions of how and when the hybridisation

events occurred. From our mtDNA sequence analysis we conclude

that the ages of the TcV and TcVI MRCAs are within

approximately the last 60,000 years. At this scale, however,

COII-ND1 provided relatively low resolution, reflected by wide95% HPD limits. The lack of allelic and genotypic diversity across

so many fast-evolving microsatellite loci and the finding that the

TcII and TcIII-derived genetic material appears unchanged,

indicates a date towards the more recent end of the mtDNA-

inferred range would be most parsimonious. Plasmodium vivaxprovides a useful comparison since it displays a similarly low level

of microsatellite diversity to that found here for TcV and TcVI

and is estimated to have expanded from a small founding

population less than 10,000 years ago [66]. When the distribution

of TcV and TcVI is considered, the data are indicative of a recent

and rapid spread of what are effectively two clonal genotypes. This

fits well the hypothesis [14] that the present distribution of TcV

and TcVI is the product of changes in the distribution of the vector

Triatoma infestans, which is itself thought to have spread from aninitial sylvatic focus in Bolivia, across the Southern cone region

and into North-eastern Brazil as a result of human-mediated

dispersal and adaptation to synanthropic and domestic niches

[67–69].

Two scenarios for TcV and TcVI origins can be considered.

Firstly, limited diversity in hybrids now found in domestic cycles

could result from a genetic bottleneck associated with invasion of

domestic niches from more diverse, unsampled TcV/VI sylvatic

populations. Records of non-domestic TcV and TcVI are,

however, extremely rare. Multiple foci of wild T. infestans occurin the Gran Chaco, Chile and especially in highland Bolivia

(reviewed in [70]), but only TcI genotypes have so far been



documented [71]. Alternatively the hybrids are anthropogenic

with human activities leading to a set of conditions initially

promoting hybridisation between TcII and TcIII and then

establishment of hybrid populations in newly available niches.

Subsequent bottlenecks may have also contributed to low TcV/VI

diversity. Humans had arrived in South America by 14,600 years

ago and possibly earlier [72]; transmission of T. cruzi to humanswas a frequent occurrence from at least 9,000 years ago as shown

by analysis of mummified specimens [73].

Co-infection of a single host or vector would have been

prerequisite for hybridisation and current distributions suggest a

domestic/synanthropic setting is more likely. Like TcV and TcVI,

TcII occurs predominantly in domestic transmission cycles [7,74–

76]. TcIII is most frequently associated with Dasypus armadillosbut also has domestic foci involving dogs and T. infestans in theGran Chaco [43,76,77]. The mtDNA sequences showed that the

most genetically similar TcIII isolates to the hybrids were isolated

mainly from domestic sources. Human activities potentially

promoting co-infection include provision of habitats favourable

for triatomine domiciliation [69] and practices associated with

hunting [73].

When the geographic, ecological and palaeontological data are

considered in the context of the extreme lack of microsatellite

diversity in TcV and TcVI and the possible timescale for their

emergence inferred from our data, we propose that anthropogenic

hybridisation events are more plausible than invasion from a

sylvatic source. TcII and TcIII are ancient lineages that would

have been well adapted to sylvatic niches and so any inter-lineage

hybrids would likely be out-competed. Therefore, an anthropo-

genic origin for the hybrids may also explain their establishment

and success, which is evident from their widespread current

distribution, association with domiciliated T. infestans and humaninfectivity. TcV in particular predominates in human infections in

Bolivia and appears to be well-adapted to congenital transmission

[78–81].

Mechanism of hybridisationT. cruzi laboratory hybrids produced to date are products of

diploid fusion followed by genome erosion [19]. The mechanism

of gene loss is incompletely characterised but does not involve a

rapid return to diploidy [20]. It is yet to be determined whether

such a mechanism operates in the field. Diploid fusion in T. cruzi isin contrast with T. brucei and Leishmania major for which laboratorycrosses normally generate diploid progeny resembling standard

meiotic F1 hybrids [82–86]. However, no haploid stages have

been observed and polyploid and/or aneuploid hybrid progeny

occur frequently [82,87,88]. There are numerous precedents for

non-meiotic tetraploid to diploid transitions, for example, in

Saccharomyces cerevisiae, as a result of repeated mitoses [89,90], or inCandida albicans, in response to stress conditions [91,92].

An expected consequence of diploid fusion followed by a

4nR2n transition is the presence of non-recombinant (non-hybrid)genotypes in 2n progeny at one third of unlinked loci, assuming

that loss of chromosomal homologues from the 4n intermediate is

random with respect to parentage, as observed in the C. albicansparasexual cycle [93]. In this context we examined the TcV and

TcVI marker data but identified only 3/28 microsatellite loci with

evidence for non-hybrid genotypes. Two of these loci were also

homozygous and two were physically linked to other loci with

hybrid, heterozygous genotypes. Therefore, LOH by gene

conversion or the presence of alleles in unsampled TcII or TcIII

strains are more likely explanations than uniparental inheritance.

Multilocus sequence analysis has now shown evidence for

relatively frequent LOH in TcV and TcVI coding sequences

[57]. Some alleles may have been wrongly classified as parental

due to homoplasy; however, the frequency of such errors is likely

to be low. For example, of 196 alleles in TcII and TcIII only 31

were found in both DTUs, showing that independent evolution

has tended to create divergent rather than convergent genotypes.

TcV and TcVI have hybrid genotypes for most markers

examined in a sufficiently representative sample [16,17,94–96].

Non-hybrid (TcII/TcII or TcIII/TcIII) genotypes have been

identified in TcV/VI for a small proportion of other markers,

including SL-RNA, 24Sa rDNA (TcVI only), and HSP70[3,17,23]. Again though, LOH better explains the absence of

one parental allele rather than differential loss during a 4nR2ntransition, particularly since these sequences all correspond to

multicopy genes, which appear more susceptible to gene

conversion [97]. The CL Brener (TcVI) genome analysis gives

additional support to our conclusions since equal proportions of its

genome are derived from TcII as from TcIII with only 3% being

classified as homozygous [98].

Thus, overall there is strong evidence that TcV and TcVI

strains do not deviate significantly from the 1:1 ratio of TcII:TcIII

alleles expected for the clonal progeny of standard meiotic F1

hybrids and so the operation of meiosis in natural cycles should not

be ruled out. Scenarios involving fusion followed by either random

loss of chromosomes (parasexual reduction) or by a meiotic

reductive division (i.e. [4nR8nR2(4n)R4(2n)]) with independentassortment of parental chromosomes, would be expected to result

in a far higher proportion of non-hybrid genotypes than is

observed. Nevertheless, the lack of an observed haploid stage and

recombination via diploid fusion in vitro suggest that the

programme of sexual reproduction in T. cruzi may differ from

canonical meiosis yet still enable production of viable diploid

recombinants, for example by meiosis with constraints on

chromosomal assortment [99] or one-step meiosis [100].

While the details of the cytological mechanism remain

somewhat unclear, the epidemiological consequences of recent

hybridisation are not: TcV and TcVI are important agents of

Chagas disease that are frequently isolated from domestic

transmission cycles in parts of South America where severe

manifestations (e.g. megasyndromes) and congenital transmission

are prevalent. It will be important to determine why the hybrids

and one of their parents are well adapted to domestic transmission

while the other parent has remained largely sylvatic. Modern

human activities continue to disrupt sylvatic T. cruzi transmission

cycles and may promote the emergence of novel, virulent

recombinant strains.

Supporting Information

Figure S1 Inferred patterns of allelic inheritance inhybrid multilocus genotypes. Unique multilocus genotypes(MLGs) for TcV (28/D01, 28/D02) and TcVI (28/E01, 28/E02,

28/E03) are shown with loci ordered in synteny based on the

position of loci in the CL Brener (TcVI) reference genome

sequence. Underlined loci contain fixed differences between TcV

and TcVI; alleles in bold italic type indicate intra-DTU genotypic

variability. Alleles are shaded according to their presence or

absence among parental DTU (TcII and TcIII) samples: yellow,

TcIII-restricted; blue, TcII-restricted; green, both TcII and TcIII;

white, neither TcII nor TcIII. Boxed genotypes indicate putative

non-hybrid genotypes.

(PDF)

Table S1 Genotypes for all samples.

(PDF)



Table S2 Microsatellite locus information.(PDF)

Table S3 Sequence diversity summary statistics.(PDF)

Table S4 Multilocus microsatellite genotypes.(PDF)

Acknowledgments

We are grateful to Hernán Carrasco, Patricio Diosque, Vera Valente,

Sebastião Valente, Michel Tibayrenc and Christian Barnabé for kindly

providing T. cruzi samples. We also thank Isabel Mauricio, Sinead

Fitzpatrick and Rania Baleela for insightful discussions and Louisa

Messenger for assistance with sequencing and suggestions on the

manuscript. We thank three anonymous reviewers for their constructive

comments.

Author Contributions

Conceived and designed the experiments: MDL MAM. Performed the

experiments: MDL MSL NA MY. Analyzed the data: MDL MSL.

Contributed reagents/materials/analysis tools: MSL NA MY MWG.

Wrote the paper: MDL MSL MY MWG MAM.

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